IMMUNOLOGICAL STUDIES ON THEHONEY BEE (APIS MELLIFERA, L.)

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GILLIAM, Martha Ann, 1940-

IMMUNOLOGICAL STUDIES ON THE HONEY BEE (APIS MELLIFERA L.).

The University of Arizona, Ph.D., 1973

Health Sciences, immunology

University Microfilms, A XEROX Company, Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.

IMMUNOLOGICAL STUDIES ON THE HONEY BEE

(APIS MELLIFERA L.)

by

Martha Ann Gilliam

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF MICROBIOLOGY AND MEDICAL TECHNOLOGY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN MICROBIOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 7 3

TI-IE UNIVERSITY OF ARIZONA

GRADUATE COLLEGE

I hereby recommend that this dissertation prepared under my

direction by Martha Ann Gill iam

entitled IMMUNOLOGICAL STUDIES ON T11L iiQiJEY liL'iJ

(APIS MLLLIFLRA L.)

be accepted as fulfilling the dissertation requirement of the

degree of Doctor of Philosophy

—i .y. ^ 7~3 Dissertation Dj-t'ector Date

i

After inspection of the final copy of the dissertation, the

following members of the Final Examination Committee concur in

its approval and recommend its acceptance:"'-'

, ^ /

(.

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

ACKNOWLEDGMENTS

I thank Dr. Wayburn S, Jeter for his encourage

ment and guidance during this investigation.

My sincere appreciation goes to the U. S. Depart

ment of Agriculture, Entomology Research Division, for

Cooperative Agreement Grant No. 12-14-100-9062(33) which

supported this work.

Dr. M. D. Levin of the U.S.D.A. Bee Research

Laboratory provided encouragement, working space, and

certain necessary equipment. Mr. Stephen Taber, III, of

the U.S.D.A. assisted with the honey bees, and to them I

am grateful.

iii

TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS vi

LIST OF TABLES vii

ABSTRACT viii

INTRODUCTION 1

MATERIALS AND METHODS 5

Vaccine Preparation and Immunizing Materials 5

B e e s . . . » 5 Mammals 6

Bee. Injection and Bleeding .......... 6 Immunization of Mammals 7 Serological Tests ..... 9

Agglutination Tests ... 9 Precipitin Tests ............. 10 Complement Fixation Tests 11

Electrophoretic Analyses 12

RESULTS It

Agglutinating Substances in Honey Bees Injected with Bacillus Larvae Vaccine 14-Comparisons of bee Anti-B. Larvae Hemo-lymph and Normal Memo lymph 15

Electrophoretic Analyses 15 Serological Analyses 15

Precipitating Substances in Honey Bees Injected with BSA 20 Comparisons of Bee Anti-BSA Hemolymph and Normal Hemolymph ..... 22

Electrophoretic Analyses . 22 Serological Analyses 22 Immunoelectrophoresis 26

DISCUSSION 30

iv

V

TABLE OF CONTENTS--Continued

Page

SUMMARY 35

LITERATURE CITED 36

LIST OF ILLUSTRATIONS

Figure Page

1. The agglutination titers of bee anti-B« larvae hemolymph as a function of •time following immunization 16

2. Electrophoretic patterns of the hemolymph from bees injected 24 hours previously with Bacillus larvae vaccine and from normal bees 17

3. The precipitin titers of bee anti-BSA hemolymph as a function of time following immunization . 21

4-. Electrophoretic patterns of the hemolymph of (a) normal bees, and (b) bees injected 24 hours previously with BSA . . 23

5. Electrophoretic patterns of (a) BSA, and (b) the in vitro mixture of BSA and bee hemolymph 24

6. Typical precipitin lines developed during IEP by the interaction of the antigen, normal honey bee hemolymph (placed in wells), and rabbit anti-BSA hemolymph serum (in trough) . 28

7. Typical precipitin lines developed during IEP by the interaction of the antigen, bee anti-BSA hemolymph (placed in wells), and rabbit anti-BSA hemolymph serum (in trough) 29

vi

LIST OF TABLES

Table Page

1. Titers of Sera from Mice Immunized with Normal Honey Bee Hemolymph and Bee Anti-13. Larvae Hemolymph 19

2. Titers of Sera from Rabbits Immunized with Normal Honey Bee Hemolymph and Bee Anti-BSA Hemolymph 25

vii

ABSTRACT

Adult worker honey bees, Apis mellifera L., were

injected with soluble and particulate antigens to deter

mine whether they could produce antibody-like substances.

Bovine serum albumin (BSA) was used as the soluble anti

gen and a vaccine prepared from Bacillus larvae was the

particulate antigen. Bees received 5 yl of the test anti

gen. Most of the bees were bled 24 hr after the injection.

However, other bees were bled at 6-hr intervals for 120 hr

after injection to determine the duration of the aggluti

nating and precipitating substances.

An agglutination titer of 1280 against B_. larvae

was observed with a bee anti-B. larvae hemolymph. Bee

anti-BSA hemolymph had a precipitin titer of 64-000 against

BSA. These titers reached their maximum levels at 2 4- hr

and then slowly declined. After 96 hr, no precipitating

or agglutinating substances were detectable. No

complement-fixing substances were found in any bees.

Injections of BSA and B_. larvae altered the pro

tein patterns of the hemolymph of bees as demonstrated by

polyacrylamide gel electrophoresis. New and different

proteins appeared which did not correspond to those of the

antigens injected.

viii

ix

To determine whether normal hemolymph and hemo

lymph from injected bees differed, mice were immunized

against normal hemolymph and bee anti-B. larvae hemolymph.

In addition, antisera against normal hemolymph and bee

anti-BSA hemolymph were prepared in rabbits. Serological

analyses revealed that only low-level cross-reactivity

existed between normal and "immune" hemolymphs.

INTRODUCTION

Literature prior to 19 3 0 on immunity in insects was

reviewed by Chorine (1) and Paillot (2), whereas literature

on immunity of invertebrates in general was surveyed by

Huff (3). Briggs (4} 5), Stephens (6), Wagner (7), and

Heimpel and Harshbarger (8) considered more recent studies.

Much controversy has existed concerning the possi

bility of antibody production in insects. Stephens (6)

stated that the demonstration of a mammalian type of anti

body response in insects seemed improbable. Glaser (9)

reported an agglutinin formed by grasshoppers in response

to an injection of Bacillus poncei, but Briggs (4) was un

successful in his attempts to find agglutinins for various

particulate antigens in the hemolymph of lepidopterous

larvae. Paillot (10) successfully immunized caterpillars

against Bacillus melolonthae-non-liquifascians by inoc

ulating the insects with a 3-month-old culture of the

organisms. Twenty-four hours after the vaccination, these

caterpillars were immune to challenge doses of the virulent

culture which were lethal to non-vaccinated controls.

Glaser (11) similarly immunized silkworms against disease

organisms by inoculating the insects with killed cultures

of bacteria.

2

Stephens (12) observed that larvae of Galleria

mellonella, the greater wax moth, acquired immunity of

Pseudomonas aeruginosa. Resistance reached its maximum

level by 24 hours. Steinhaus (13, p. 213) stated, "Perhaps

the most startling thing concerned with active immunization

of insects is the fact that they can be immunized in so

short a time—usually within 2 4 hours following a single

injection of an old culture or a vaccine."

Briggs (4) concluded that the precipitin test was

not satisfactory for the demonstration of natural or

actively acquired immune substances in the hemolymph of

lepidopterous larvae. He did observe precipitins after

injection of the larvae with egg albumin. Unfortunately,

however, precipitin formation also occurred in the controls

in many instances. Fredericq (14) injected silkworm lar

vae with egg albumin and was unable to detect antibodies

by the precipitin technique. Recently, the synthesis of

antibodies against bovine serum albumin by the scorpion,

Androctonus australis, was reported (15).

Naturally occurring hemagglutinins to human eryth

rocytes were reported by Bernheimer (16) in a large

number of lepidopterous larvae. He suggested that the

agglutinins may be associated with parasitism of the lar

vae by other insects. In a subsequent communication,

Bernheimer et al. (17) demonstrated the ability of one

3

species of Lepidoptera to acquire a hemagglutinin in re

sponse to injections of human erythrocytes or egg albumin^

Complement has not been demonstrated in any inver

tebrate. Morgun (18) reported the absence of complement

in the hemolymph of several insects. However, he found

that the hemolymph of cockroaches, caterpillars, and

snails could reconstitute frog complement which is de

prived of C'3.

Bee diseases cause losses of millions of dollars

annually. In addition to destruction of colonies, pro

duction of honey and beeswax, and pollination of crops

are seriously affected. Many crops of economic impor

tance such as alfalfa, almonds, apples, and citrus are

dependent upon honey bees for pollination.

American foulbrood disease, caused by Bacillus

larvae, occurs throughout the world. It is a serious

disease, killing not only large numbers of individual bees

but also destroying colonies. It is the disease most

feared by beekeepers since it is always a potential men

ace when protective measures are slackened.

Gary et al. (19) reported that B. larvae cells

were agglutinated by the hemolymph of honey bees from a

colony infected with American foulbrood. Toumanoff (20)

found that vaccinated adult honey bees were less resistant

to infection with Bacillus alvei if the interval of the

4

challenge was increased from one to five days. Only half

of the surviving bees were still refractory to rechallenge

six days later.

The purpose of this investigation was to determine

whether honey bees, Apis mellifera L., are capable of pro

ducing antibody-like substances in response to injections

of soluble and particulate antigens and to determine by

serological and immunological methods wliether normal nemo-

lymph and hemolymph from "immune" bees differs.

MATERIALS AND METHODS

Vaccine Preparation and Immunizing Materials'

Bees

A culture of B. larvae was obtained from Dr.

Hachiro Shimanuki of the U.S.D.A. Bee Disease Laboratory

in Beltsville, Maryland. The organisms were grown in

brain heart infusion broth (Difco) supplemented with 0.01%

thiamine hydrochloride for 72 hr at 37 C. An equal volume

of 0.6% formalin in 0.85% NaCl was added to the culture

and allowed to stand at room temperature for 3 days. After

confirming the bacterial sterility of the culture, the or

ganisms were sedimented by centrifugation at 150 0 x G for

30 minutes. Subsequently, the supernatant fluid was re

moved, and the bacteria were resuspended in 100 ml of 0.3%

formalinized saline. Prior to use, the vaccine was di

luted with sterile 0.85% saline by comparison with a O

nephelometric standard to contain 12 x 10 organisms (both

spores and vegetative cells) per ml. Unfortunately, no

definitive studies on the ionic composition of bee hemo-

lymph have been reported. Therefore, 0.85% saline has been

universally accepted as a diluent for bees. Vaccines of

5

6

Salmonella thompson and Bacillus subtilis were prepared in

the same manner.

Bovine serum albumin (BSA) and human serum albumin

(HSA) were purchased from the Pentex Corporation, Kankakee,

Illinois. A 1% solution of BSA prepared in 0.85% NaCl was

used for injection of bees.

Mammals

Bee anti-B. larvae hemolymph obtained from bees in

jected with 13. larvae vaccine was used to immunize mice,

and bee anti-BSA hemolymph from bees injected with 1% BSA

was used for immunization of rabbits. Cell-free normal

hemolymph from unimmunized bees was used for control

immunizations.

Bee Injection and Bleeding

Adult worker honey bees were anesthetized with

carbon dioxide and injected individually with 5 Pi of the

antigen (either 1% BSA or B. larvae vaccine). Five y1 is

the largest amount of solution which can be injected with

out leakage. Injections were made dorsally in the

thoracic hemocoel using a microliter syringe and a 27-

gauge needle. Control bees received 5 yl of 0.8 5% saline,

carbon dioxide only, a stab in the thoracic hemocoel with

a 2 7-gauge needle, or no treatment. Each group contained

approximately 2 00 bees. Twenty-four hr later, the bees

were again anesthetized with carbon dioxide. Most of the

insects were decapitated with a scalpel, and the drop of

hemolymph which exuded from the thorax was collected with

a capillary pipette. Approximately 1 - 3 yl of hemolymph

was obtained from each bee.

All hemolymph was centrifuged under refrigeration

at 2500 x G for 20 min to remove the hemocytes. The hemo

lymph from each group of bees was pooled in acid-washed

vials and stored at -70 C prior to use.

The site of injection and the body region from

which the blood was drawn were varied in some experiments.

The bees were either injected dorsally through the mem

brane between the third and fourth abdominal segments and

then bled from the thorax, or they were injected in the

thorax and bled from the abdomen.

Large groups of bees were also injected with

either BSA or B_. larvae vaccine and bled at B-hr intervals

after injection for 120 hr to determine the effect of time

on the titers of immune substances.

Immunization of Mammals

Bee anti-B. larvae hemolymph which had an aggluti

nation titer of 12 8 0 against the original B. larvae

vaccine was injected into 28 BALB/c 3-month-old male mice

which were obtained from departmental colonies. Alsc, 28

mice were injected in the same manner with normal

hemolymph. Each mouse was given 2 subcutaneous injections

10 days apart, each containing 0.04 ml of hemolymph (1 mg

protein) in 0.0 4 ml incomplete Freund's adjuvant (21). The

mice were maintained on Purina mouse food and water. Ten

days after the second injection, all mice were exsangui

nated, and the serum was frozen at -7 0 C until used.

Cell-free normal hemolymph obtained from unimmu

nized bees was used for control immunization of rabbits.

Protein estimations were performed on this normal hemolymph

using 280 nm/260 nm spectrophotometry ratios (22). Since

normal hemolymph contained approximately 2 5 mg of protein

per ml, all hemolymph used for injection was adjusted to

this concentration with sterile 0.85% NaCl.

Eight New Zealand male albino rabbits weighing be

tween 2,5-4.0 kg were test bled from the ear vein. Two

groups of 4 rabbits each were immunized, one group with

normal hemolymph and the second group with anti-BSA hemo

lymph which had a precipitin titer of 64000. In addition,

2 rabbits were injected with 1% BSA. The animals were in

jected with an emulsion prepared with equal parts of the

antigen solution (hemolymph or BSA) and Freund's incom

plete adjuvant (21). For each injection, a total of 2 ml

of the emulsion were injected intradermally into 2 0 sites

(0.1 ml each) in the shaven back of each rabbit. Ten days

later the injections were repeated in each of the rabbits.

9

The rabbits were maintained on Purina pellets and water ad

libitum. Eight weeks after the second injection, the

rabbits were exsanguinated by heart puncture. The serum

was collected and stored at -70 C until used.

Serological Tests

Agglutination Tests

Beginning with a 1:10 dilution of antibody, 2-fold

dilutions to 1:2560 were prepared in 0.8 5% NaCl. One ml of

the antigen was added to 1 ml of each antibody dilution in

a test tube. Saline replaced either the antigen or anti

body for controls. The tubes were incubated at 37 C in a

water bath for 18 hr and observed for agglutination.

In the first series of tests, bee anti-_B. larvae

hemolymph was the antibody and B_. larvae vaccine the anti

gen. A duplicate set of tubes was incubated at 4 C. Also,

to test for nonspecific cross-reactivity of bee anti-B.

larvae hemolymph with antigenically unrelated organisms,

Salmonella thompson vaccine was employed as the antigen.

To show specificity with a related bacterium, a Bacillus

subtilis cellular suspension was used.

In the second series of tests, mouse anti-B. larvae

hemolymph was the antibody and either B. larvae vaccine or

bee anti-B. larvae hemolymph the antigen. Bee anti-B.

larvae hemolymph was used as the antigen to determine

10

whether this hemolymph contained residual B. larvae cells

which could be transferred to mice by injections of hemo

lymph. B. larvae vaccine was used as an antigen to detect

antibodies to 13, larvae produced by mice which had been

immunized with bee anti-B. larvae hemolymph. Mouse anti-

hemolymph serum tested with B. larvae vaccine as the

antigen served as a control.

Precipitin Tests

The ring or interfacial test was employed. The

following antigen dilutions were prepared using saline as

the diluent: 1:10; 1:100; 1:1000; 1:2000; 1:4000; 1:8000;

1:16000; 1:32000; 1:64000; 1:128000; and 1:256000.. The

antibody was first placed in 4 x 50 mm acid-washed tubes

and was then layered with each antigen dilution. Saline

replaced either the antigen or antiserum for controls. All

tubes were examined after 30, 60, and 90 minutes, and the

titer was recorded as the reciprocal of the highest di

lution of antigen giving a positive reaction.

Bee anti-BSA hemolymph was the antibody and 1% BSA

the antigen in the first series of tests. Hemolymph from

control groups of bees was also tested against 1% BSA. To

test the specificity of bee anti-BSA hemolymph, human

serum albumin (HSA) was used as.the antigen in precipitin

tests.

11

The precipitin titers of antisera from mice immu

nized with normal hemolymph and bee anti-B. larvae

hemolymph were determined against normal hemolymph, con

trol hemolymph (stabs, CO2 treatment, saline injection),

and bee anti-B_. larvae hemolymph. Normal mouse serum was

used as an antibody control against normal hemolymph and

bee anti-B. larvae hemolymph.

The precipitin titer of the antiserum from each

rabbit immunized with normal or bee anti-BSA hemolymph was

determined against normal hemolymph, anti-BSA hemolymph,

and 1% BSA. Rabbit anti-BSA serum was tested against 1%

BSA and bee anti-BSA hemolymph to detect residual BSA in

the hemolymph. In addition, two-dimensional gel diffusion

tests were performed using Immuno-plates^ from Hyland

Laboratories. The antibody was placed in the center well

and the antigen dilutions in the outer wells. The plates

were prepared in triplicate. One set was incubated at 4 C

one at 25 C, and another at 37 C for 10 days.

Complement Fixation Tests

These tests were performed according to the method

of Campbell et al. (23) using fresh guinea pig serum as

the source of complement. Bee anti-B_. larvae hemolymph

and bee anti-BSA hemolymph were analyzed for complement-

fixing substances against their homologous antigens.

12

Electrophoretic Analyses

Freshly collected blood from control bees, bees in

jected with 13. larvae vaccine, and bees injected with BSA

was subjected to separation by a Canalco Model 12®poly-

acrylamide gel disc electrophoresis apparatus (24, 25).

Reagents were obtained from Canalco, Rockville, Maryland,

Each analysis was carried out at 5 ma per tube with a pH

8.5 tris-glycine buffer with an ionic strength of 0.01.

Previous experiments had shown that hemocytes were trapped

in the stacking gel. Centrifuged and non-centrifuged hemo-

lymph yielded identical electrophoretic patterns. There

fore, blood used in these experiments was not centrifuged.

The gels were stained with Amido-Schwartz, electrophoreti-

cally destained and stored in 7.5% acetic acid (26), and

examined for differences in protein patterns. In addition,

all gels were analyzed with a Photovolt® densitometer and

automatic integrator. 13. larvae vaccine, BSA, and in

vitro mixtures of hemolymph plus vaccine or BSA (1:1, 1:2,

2:1) were separated.

Immunoelectrophoresis of these rabbit sera was per

formed using the Gelman apparatus. The technique outlined

in the Gelman Manual was followed,which is a modification

of the method of Scheidegger (27). Veronal buffer pH 8.6

with an ionic strength of 0.1 was employed. Current

(10 ma/frame) was applied for one hour. After addition of

the antiserum, the .inununoprecipitate contained in the agar

gel was stained with Amido-Schwartz dye.

RESULTS

Agglutinating Substances in Honey Bees Injected witn bacillus

Larvae Vaccine

The first studies were concerned with agglutinins

against Bacillus larvae produced by honey bees. An agglu

tination titer of 1280 was observed with bee anti-B_.

larvae hemolymph. Bees injected with 0.8 5% saline,

treated witn carbon dioxide, stabbed in the thorax with a

27-gauge needle, or receiving no treatment produced no

agglutinins to B_. larvae. All saline controls were nega

tive. The entire experiment (injection of bees, bleeding

of bees, and agglutination tests) was repeated ten times,

and the same titer was obtained in each case. The same re

sults were observed regardless of the injection site or the

body region from which the blood was drawn. These findings

indicate that agglutinating substances are produced and

disseminated rapidly within the body of the honey bee. No

agglutination was observed when S_. thompson, an antigeni-

cally unrelated organism, was employed as the antigen in

the agglutination test. On the other hand, an aggluti

nation titer of 20 was observed when B. subtilis, a species

related to B. larvae was used. The same results were

14

15

obtained whether the tubes were incubated at 37 C or at 4 C.

However, 24-30 hr were required for agglutination to occur

at 4 C. No complement-fixing substances were detected.

The agglutination titer reached its maximum level

at 24 hr after immunization and then slowly declined. After

9 6 hr, no titer was detectable (Fig. 1).

Comparisons of Bee Anti-B. Larvae He mo lymph and Normal liemo'lymph

Electrophoretic Analyses

Injections of B. larvae vaccine altered the protein

electrophoretic pattern of bee hemolymph (Fig. 2). Elec

trophoretic patterns from all groups of control bees were

identical. None of the protein bands of bee anti-B. larvae

hemolymph was similar to those present in the vaccine

alone. Also, when varying proportions of normal hemolymph

and vaccine were mixed in vitro and then subjected to elec

trophoresis, the mixtures did not contain bands

corresponding to "immune" hemolymph.

Serological Analyses

These experiments were performed to determine

whether normal hemolymph and bee anti-B. larvae hemolymph

differ. The results of the agglutination and precipitin

tests of antisera from mice injected with normal hemolymph

and mice injected with bee anti-B. larvae hemolymph are

5120-1

2560-

1280-

cc 640-LlI — 320-

160-

80-

Z>

40-

20-

10-

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120

TIME IN HOURS

Fig. 1. The agglutination titers of bee anti-B. larvae hemolymph as a function of time following immunization

cn

17

Fig. 2. Electrophoretic patterns of the hemolymph from bees injected 24 hours previously with Bacillus larvae vaccine and from normal bees

18

shown in Table 1. Mouse anti-13. larvae hemolymph serum

gave an agglutination titer of 40 against B_. larvae vac

cine and an agglutination titer of 10 against bee anti-13.

larvae hemolymph. Ho agglutination titer was observed

when mouse anti-hemolymph serum was used as the antibody.

These tests indicated that some vaccine particles were not

changed in the bees and that these particles were in

jected into the mice which then produced antibodies

against 13. larvae vaccine.

Mouse anti-B. larvae hemolymph serum had a precip

itin titer of 2000 against bee anti-B^ larvae hemolymph,

the homologous antigen. It had titers of 10 and 10 0

against control hemolymphs which indicates low-level cross-

reactivity between normal and "immune" hemolymphs. Mouse

anti-hemolymph serum showed a precipitin titer of 4 00 0

against normal hemolymph and all control hemolymphs and a

titer of 10 against bee anti-13. larvae hemolymph, which

also shows some cross-reactivity between the two hemo

lymphs .

No precipitation occurred when normal mouse serum

was tested against normal hemolymph or bee anti-B. larvae

hemolymph. These results indicated that nonspecific pre

cipitin reactions did not occur.

19

Table 1. Titers of Sera from Mice Immunized with IJormal Honey Bee Hemolymph and Bee Anti-B. Larvae Hemolymph

Antibody

Agglutination

Antigen Titer

Precipitin Titer

Mouse anti-B. larvae hemolymph serum

B. larvae vaccine 40

Mouse anti-B. larvae hemolymph serum

Bee anti~B. larvae hemolymph 10 2 00 0

Mouse anti-B. larvae hemolymph serum

Normal hemolymph 100

Mouse anti-B. larvae hemolymph serum

Control hemolymph (Stabs, C02 treatment only; 10

Mouse anti-B. larvae hemolymph serum

Control hemolymph (Saline injection) 100

Mouse anti-hemolymph serum

B. larvae vaccine 0

Mouse anti-hemolymph serum

Bee anti-B. larvae hemolymph 10

Mouse anti-hemolymph serum

Normal hemolymph 4000

Mouse anti-hemolymph serum

Control hemolymph (Stabs, CO2 treatment only, saline inj ection) 4000

Normal mouse serum Normal hemolymph 0

Normal mouse serum Bee anti-B. larvae hemolympFT 0

20

Precipitating Substances in Honey Bees ~Trijecte-cr"witn BSA

A precipitin titer of 64000 against BSA was ob

served with bee anti-BSA hemolymph. The entire experiment

from injection of bees through precipitin tests was re

peated five times, and the same titer was obtained in each

case. All controls were negative. The same results were

obtained regardless of the injection site or the body

region from which the blood was drawn. The titer reached

its maximum level at 2 4 hr and then slowly declined (Fig.

3). After 96 hr, no precipitating substances were detect

able. A precipitin titer of 8 000 was observed when USA was

used as the antigen against bee anti-BSA hemolymph. How

ever, no reaction occurred with HSA and normal hemolymph.

No reaction was observed when rabbit anti-BSA serum was

used as the antibody against bee anti-BSA hemolymph, sug

gesting that no residual BSA molecules remained in bee

anti-BSA hemolymph.

Single precipitin bands of identity were visible

after incubation for 2 4 hr at 2 5 C and 37 C on Immuno-

plates containing bee anti-BSA hemolymph as the test

substance at all BSA antigen dilutions through 16000 and

also at all HSA dilutions through 1000. No bands were

observed when the hemolymph of control bees was used.

Plates incubated at 4 C required 48 hr for the bands to

64,000-i

32,000-

16,000-

Q: £ 8000-

I-4000-

100-

10-

0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120

TIME IN HOURS

Fig. 3. The precipitin titers of bee anti-BSA hemolymph as a function of time following immunization

22

appear. No differences in bands were observed on plates

incubated at different temperatures, and no additional

bands appeared after longer incubation. No complement-

fixing substances for BSA were detected in the bees.

Comparisons of Bee Anti-BSA Hemolymph and Normal iiemolympTT

Electrophoretic Analysis

The electrophoretic patterns of hemolymph from bees

injected with BSA differed from those of untreated bees

(Fig. 4). The gels from these two groups of bees contained

few, if any, corresponding bands. Normal hemolymph con

tained 9 protein baxids , while hemolymph from bees injected

with BSA yielded 16 bands. These additional protein bands

were not found when BSA and hemolymph were mixed in vitro

and analyzed (Fig. 5). In addition, no hemolymph bands

corresponded to the BSA bands when BSA alone was electro-

phoretically separated. Blood from all control groups of

bees gave electrophoretic patterns identical to that of

untreated bees.

Serological Analyses

These tests were performed to determine similar

ities or differences in normal hemolymph and bee anti-BSA

hemolymph. The results of the precipitin tests are shown

in Table 2.

ORIGIN OHIO IN I I

FRONT M I HON I 1*1

(a) (b)

Fig. 4. Electrophoretic patterns of the hemolymph (a) normal bees, and (b) bees injected 24 hours previously with BSA

-V6

MM

•

(a) (b)

Fig. 5. Electropnoretic mixture

patterns of (a) BSA, and of BSA and bee hemolymph

(b) the in vitro

ro -P

25

Table 2. Titers of Sera from Rabbits Immunized with Normal Honey Bee Hemolymph and

Bee Anti-BSA Hemolymph

Antibody Antigen

Tube Precipitin Titer

Gel Diffusion Precipitin Titer

Rabbit anti-hemolymph serum

Normal hemolymph 61+000 8000

Rabbit anti-hemolymph serum

Bee anti-BSA hemolymph 4000 10

Rabbit anti-hemolymph serum

1% BSA 0 0

Rabbit anti-BSA hemolymph serum

Normal hemolymph 4000 100

Rabbit anti-BSA hemolymph serum

Bee anti-BSA hemolymph 16000 4000

Rabbit anti-BSA . hemolymph serum

1% BSA 16000 1000

Rabbit anti-BSA serum

1% BSA 8000 1000

Rabbit anti-BSA serum

Bee anti-BSA hemolymph 0 0

26

All rabbit sera collected prior to immunization had

no. detectable precipitin titers against normal hemolymph,

bee anti-BSA hemolymph, or BSA. Rabbit anti-hemolymph

serum gave a precipitin titer of 64000 against normal hemo

lymph, indicating that bee hemolymph is an excellent antigen,

however, rabbit anti-hemolymph serum had a precipitin titer

(4000) against bee anti-BSA hemolymph. This observation in

dicated that only low-level cross-reactivity existed between

normal and "immune" hemolympn but that these hemolymphs con

tained some common components. Rabbit anti-hemolymph serum

contained no,, precipitins against 1% BSA.

Rabbit anti-BSA hemolymph serum contained precip

itins against anti-BSA hemolymph in a titer of 16000. In

addition, rabbit anti-BSA hemolymph serum had a precipitin

titer of 4000 against normal hemolymph, indicating low-level

cross-reactivity between normal and "immune" hemolymph.

Since rabbit anti-BSA hemolymph serum had a titer

of 16000 against 1% BSA, rabbit anti-BSA serum was used as

the antibody in precipitin tests to detect unchanged bSA

in bee anti-BSA hemolymph. ilo BSA was found in the hemo

lymph by this method.

Immunoelectrophores is

Immunoelectrophoresis revealed at least two addi

tional bands in rabbit anti-BSA hemolymph serum when bee

anti-BSA hemolymph was used as the antigen (Figs. 6 and 7).

These results indicate differences between the two hemo-

lymphs.

Fig. 6. Typical precipitin lines developed during IEP by the interaction of the antigen, normal honey bee hemolymph (placed in wells), and rabbit anti-BSA hemolymph serum (in trough) CD

Fig. 7. Typical precipitin lines developed during IEP by the interaction of the antigen, bee anti-BSA hemolymph (placed in wells), and rabbit anti-BSA hemolymph serum (in trough)

ro CD

DISCUSSION

An agglutination titer of 1280 against B. larvae

vaccine was found in the hemolymph of bees injected with

the homologous vaccine. The titer1 reached its maximum

level at 2 4- hr and slowly declined. After 9 6 hr, no titer

was detectable. These same results were obtained when the

site of injection and body region from which the hemolymph

was drawn were varied* Therefore, agglutinating sub

stances were produced and disseminated rapidly within the

body of the honey bee. Control determinations using

either S_. thompson or B. subtilis as the test antigen in

dicated that the agglutinating materials possessed

specificity for B. larvae.

To detect differences between normal hemolymph and

bee anti-B. larvae hemolymph, antisera against each kind

of hemolymph were prepared in mice. House anti-B. larvae

hemolymph serum gave an agglutination titer of 40 against

B. larvae vaccine. This antiserum also showed a titer of

10 against bee anti-iS. larvae hemolymph, which indicated

residual B. larvae organisms in this hemolymph. There

fore, some unaltered vaccine particles were injected into

mice immunized with bee anti-B. larvae hemolymph, and tne

mice could then have produced antibodies against the

30

31

unaltered particles. When this hemolymph was centrifuged

to remove the hemocytes, stained by the gram method, and

then observed microscopically, a few distorted gram posi

tive rods were observed.

Electrophoresis of hemolymphs on polyaery1amide

gel demonstrated that injections of 13. larvae vaccine com

pletely altered the protein pattern of bee hemolymph,

indicating that the hemolymph was changed after vacci

nation. In addition, mouse anti-B. larvae hemolymph serum

had a precipitin titer of 20 00 against bee anti-B. larvae

hemolymph, the homologous antigen. It only showed a titer

of 100 against normal hemolymph. On the other hand, mouse

anti-hemolymph serum had a precipitin titer of 4000 against

normal hemolymph and a titer of 10 against bee anti-13.

larvae hemolymph. All these data strongly support a change

in the hemolymph following immunization. However, some

low-level cross-reactivity did exist between the two hemo-

lymphs, which suggested that not all proteins were altered.

A new specificity existed in bee anti-J3. larvae hemolymph

which was not present in normal hemolymph.

A similar situation was obtained when BSA, a molec

ular antigen, was used as the immunizing substance, A

precipitin titer of 64000 against BSA was observed with bee

anti-BSA hemolymph. It reached a maximum at 24 hr, and

after 96 hr, again no titer was detectable. The same

results were obtained when the sites of injection and

bleeding were varied. Therefore, precipitating substances

are also produced and disseminated rapidly within the body

of the adult honey bee. Low-level cross-reactivity between

HSA and BSA occurred in the bee as it does in higher forms

(28). Since no reaction was observed when rabbit anti-BSA

serum was tested against bee anti-BSA heinolymph as the

antigen, the BSA would appear to be altered in some manner

so that it was no longer detectable by the homologous anti

body.

Also, in bees immunized against BSA, the hemolymph

was found to have no electrophoretic bands in polyacryla-

mide gel which corresponded to the bands given by BSA. Bee

anti-BSA hemolymph contained 7 bands which were not found

in normal hemolymph. Therefore, hemolymph was altered

after injection of BSA. The BSA might have been degraded,

metabolized, altered in physical properties, changed in

chemical properties, or conjugated chemically to substances

present in bees which would alter the electrophoretic

mobility.

Immunization of rabbits with normal and anti-BSA

hemolymph and analyses of the antisera produced indicated

that only low-level cross-reactivity existed between normal

and "immune" hemolymph but that these hemolymphs contained

some common components.

33

However, rabbit anti-BSA hemolymph serum contained

precipitins against the homologous antigen and also against

1% BSA, This is contradictory to the observations with bee

anti-BSA hemolymph. This suggests that BSA was altered as

an antigen in serological situations but that it retained

the integrity of its determinant groups sufficiently to act

as an immunogen. This could alternatively be a question of

amounts of material that are required for antigenic func

tion being greater than that for immunogenic activity.

Also, at least two additional bands were revealed in rabbit

anti-BSA hemolymph serum when bee anti-BSA hemolymph was the

antigen in immunoelectrophoretic analysis as compared to

normal bee hemolymph being used as the antigen. When BSA

was used as the antigen, no bands corresponding to the bee

anti-BSA hemolymph bands were observed.

Perhaps the earlier work concerning precipitin

formation in insects was not successful because larvae

rather than adult insects were used in the experiments. It

is possible that these larvae were not immunologically com

petent. Unfortunately, larval honey bees cannot be

injected because they lack a clotting system and bleed to

death after being punctured (29).

Ho substances which could fix guinea pig comple

ment were detected in any bees, although precipitating and

agglutinating materials were present in the hemolymph of

bees injected with both soluble and particulate antigens.

It is possible that bees contain substances which might fix

the complement of other animal species. This is unlikely,

however, based on knowledge of other complement-fixing

systems, but tests using complement from other animal

species were not performed. Therefore, these results are

in agreement with those of Morgun (18).

From the results of this study, it appears that

bees are capable of producing antibody-like substances in

response to injections of antigens. After injection, bee

hemolymph contains new and different proteins. This hemo-

lymph might have been changed because of metabolic alter

ation of proteins or bonding between antigens and proteins.

However, the latter explanation seems unlikely since some

unaltered B. larvae cells regained in the hemolymph of bees

injected with the vaccine. Also, hemolymph proteins might

be altered when the immune mechanism becomes activated.

Regardless of the reason for the observed changes, the

alterations in protein patterns are striking.

SUMMARY

Adult worker honey bees, Apis mellifera L., pro

duced agglutinating substances in response to an injection

of a vaccine prepared from Bacillus larvae, the causative

organism of American foulbrood (AFB). They also produced

precipitating substances in response to an injection of

bovine serum albumin (BSA). Injections of BSA or B.

larvae produced electrophoretic changes in bee hemolymph

protein. The titers of agglutinating and precipitating

substances reached their maximum levels at 24 hr and then

slowly declined. After 96 hr, no titers were detected.

Analyses of antisera from mice and rabbits immu

nized with hemolymph from bees injected with BSA, B.

larvae, or normal hemolymph demonstrated that only low-

level cross-reactivity existed between normal and "immune"

hemolymphs.

35

/ /

S ' / s

s

A 'S

*

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36

37

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38

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